The long-term objective of the proposed research is to define the role of cell-extracellular matrix ECM) interactions in differentiation of cartilage, muscle, and nerve in their proper spatial relationships luring embryonic limb development. Recently, a recessive lethal insertional mutation in the Cspg2 gene was identified in a transgenic mouse line, hdf (heart defect). Cspg2 encodes the core protein of the chondroitin sulfate proteoglycan, versican, and hdf mutation results in loss of expression of the mature proteoglycan. Versican has been suggested to play a role in chondrogenesis and cellular patterning. However, little is known regarding details of versican function in the embryonic ECM. The hdf mutation offers a unique opportunity to explore versican function in the limb. The specific aims of this proposal are to first analyze the spatial and temporal expression of versican during limb development in the hdf mouse. LacZ reporter histochemistry, in situ hybridization, and immunohistochemistry will be utilized to determine versican expression during limb development in carefully staged hemizygous hdf and wildtype mice to correlate localization with its hypothesized functions. Second, the role of the mature versican proteoglycan in precartilage aggregation during limb chondrogenesis in vitro will be evaluated. micromass cultures of hdf limb mesenchyme will be utilized to determine if this mutation results in inhibition of prechondrogenic mesenchymal condensation and subsequent cartilage differentiation.